Serologic testing remains the most widely-used laboratory tool to support the diagnosis of Lyme borreliosis (LB), colloquially known as Lyme disease. This approach works well for some clinical applications but has major limitations in others. A specific IgM and/or IgG antibody response against the causative bacterium, Borrelia burgdorferi, may persist for decades after infection despite effective treatment. Therefore, serologic testing cannot differentiate between active and past LB, and cannot be used as a test of cure in patients whose symptoms persist despite therapy. The most well-known limitation of serologic testing, however, is its poor sensitivity in early infection. Despite the importance of diagnosis and treatment of LB in its earliest (localized) phase to prevent dissemination and subsequent development of severe and sometimes permanent pathology later in the course of infection, serologic testing is falsely negative in 70-80% of patients who present with localized erythema migrans (EM), the rash of early LB. When a classic bull's eye EM rash presents in appropriate epidemiologic context, the diagnosis is readily made, but this represents only 10-20% of EM cases. More typically, EM lesions can be difficult to distinguish from rashes caused by other infectious and non-infectious diseases; moreover, a skin rash is completely absent in approximately 20% of early LB cases. Notably, the limitations described above could potentially all be addressed by a test that can directly detect the infectious agent, rather than detecting the host's immune response to that agent. We propose to lay the groundwork for development of a novel and highly accurate test for early LB by discovering and validating novel B. burgdorferi protein biomarkers in the urine of patients with early LB. Using proven mass spectrometric (MS) methods previously applied to the discovery of novel diagnostic biomarkers of active tuberculosis (TB) and visceral leishmaniasis (VL) by the investigators, our team has identified a promising candidate B. burgdorferi biomarker in urine samples from patients with confirmed EM. We now propose to build on this promising preliminary finding by performing MS on an additional 20 urine samples from adult and pediatric patients with confirmed EM, allowing us to select the top biomarker candidates for development of antigen detection assays using methods vetted in our TB and VL biomarker work. We will then validate the diagnostic utility of the biomarker candidates by testing a large panel of urine samples from adult and pediatric patients with and without confirmed early LB. We anticipate that this work will provide firm proof of concept that B. burgdorferi protein antigens can be detected in the urine of patients with early LB, thus laying a solid foundation upon which to build in subsequent assay optimization and large-scale validation studies. We anticipate that our collaboration will lead to a substantial advance in the Lyme diagnostics field.